The measurement of blood lipoproteins is critical in predicting an individual's risk of many chronic diseases, particularly cardiovascular disease such as coronary heart disease (CHD). CHD continues to be the leading cause of death in the United States despite phenomenal advances made in its diagnosis, treatment, and prevention in the last 3 decades. As per the recently released Heart and Stroke Statistics (2012 Update by the American Heart Association; Circulation 2012; 125:e2-e220), CHD accounts for 1 in 6 deaths in the US. In 2008 as many as 405,309 people died of CHD and 785,000 were expected to have a new heart attack and another 470,000 people with recurrent attacks. These astounding statistics clearly tell us that prevention of heart disease still remains a formidable task.
Heart disease is a multi-factorial disease and several risk factors such as high blood pressure, smoking, elevated serum low density lipoprotein (LDL) cholesterol, and diabetes are attributed to an increased risk. Among these risk factors, LDL is known to be directly responsible for the build-up of the plaque within the arterial wall which results in subsequent coronary events. This is further supported by the fact that lowering LDL cholesterol by pharmacological means or lifestyle changes significantly reduces coronary events. However, only 50% of coronary events can be accounted by elevated LDL cholesterol and many studies suggest that coronary events can also occur even in people with normal LDL cholesterol. Therefore, in recent years there has been a surge in research in identifying new risk factors and biomarkers that may explain the CHD risk that cannot not be accounted by traditional risk factors. Some examples of emerging risk factors are high sensitivity C-Reactive Protein (hs-CRP), homocysteine, lipoproteins other than LDL cholesterol, such as low levels of high density lipoproteins (HDL) and its subclasses HDL2 and HDL3, and non-HDL cholesterol which includes intermediate density lipoproteins (IDL), very low density lipoproteins (VLDL), and lipoprotein(a) [Lp(a)] in addition to LDL cholesterol. Several other studies also support measurement of apolipoproteins, the proteins on the surface of lipoprotein particles. In particular, evidence to measure serum apolipoprotein B (apo B) is compelling since all atherogenic lipoproteins (Lp(a), LDL, IDL and VLDL) contain apo B and thus truly reflects the comprehensive risk associated with all atherogenic lipoproteins. In addition, serum apolipoprotein concentration also reflects the total number of atherogenic particles, which are responsible for the plaque build-up, because each of these contain one and only one molecule of apo B.
More recent studies also suggest that LDL particle (LDLp) concentration (or number) is also an independent risk marker and is superior to the risk predicted by routinely measured LDL cholesterol (more often calculated using Friedewald equation in most labs). A recent study suggests that LDL particles, not the amount of cholesterol carried by them, play a pivotal role in the development of atherosclerosis. It appears that endothelial retention of intact apo B containing particles is essential for initiation of atherosclerotic process. Thus, cholesterol in LDL molecules merely acts as ‘passenger’ while particles act as the ‘driver’. A number of published outcome studies, which used LDL particle number measurement by nuclear magnetic resonance (NMR), suggest LDL particle number is a significant and independent predictor of cardiovascular endpoints, including CHD death and myocardial infarction. Most of these studies also have demonstrated that the risk associated with elevated LDL particle number is much higher than that associated with LDL cholesterol.
As mentioned above there is sufficient evidence that increased numbers of lipoproteins other than LDL, such as elevated levels of atherogenic Lp(a), IDL, and VLDL, and low levels of anti-atherogenic HDL, are also strongly and independently associated with CHD. Thus, based on the observed clinical benefits of LDL particle concentration measurement over LDL cholesterol the measurement of particle concentration (or number) of other lipoproteins such as Lp(a), IDL, VLDL, and HDL would also result in clinical benefit and thus diagnosis and management of heart disease.
Even though an independent association of LDL particle number (LDLp) is known, LDLp number (as well as the particle number of other lipoproteins) is not commonly measured because of the following reasons. First, measurement of cholesterol is relatively easy since several simple enzymatic methods are available. Second, LDL cholesterol can be conveniently calculated using Friedewald equation [LDL−C=Total Cholesterol−(HDL cholesterol+0.2*triglycerides)]. Third, methods to measure lipoprotein particle number, including LDLp, are very few, not widely available and are complicated and expensive. The three currently available commercial assays for LDLp are based upon 1) NMR (LipoScience, NC); 2) ion mobility (Quest Diagnostics, CA); and 3) ultracentrifugation with fluorescence detection (Spectracell, TX). Furthermore, the above measurement methods do not measure the particle number of all lipoprotein classes. As a result, the particle number measurement methods cannot meet the demand for this widely required test.
Consequently, there is a long-felt but unmet need in the art to develop newer, simpler, and more accurate methods for the measurement of lipoprotein particle number, including, but not limited to, HDL, Lp(a), LDL, IDL and VLDL particle number. Particularly, there is a need to develop newer, simpler, and more accurate methods the measurement of lipoprotein particle number, including, but not limited to, HDL, Lp(a), LDL, IDL and VLDL particle number, which can be performed inexpensively in the clinical context, and which has the ability to enumerate particles of all significant types of lipoprotein. The present disclosure addresses such needs.